Method for purifying immunoglobulin solutions

ABSTRACT

Herein is reported a method for purifying cell cultivation supernatants either directly after fermentation or after one or more preliminary purification steps, such as protein A affinity chromatography. By adjusting the pH value in the acid range and subsequent incubation of the acidified solution host cell nucleic acid and host cell protein can be precipitated but the target polypeptide remains in solution. Thereafter the precipitate and therewith the contaminating host cell components can be removed by a simple physical separation step.

Herein is reported a method for removing host cell DNA and host cell protein from solutions, such as cell cultivation supernatants or protein A chromatography eluates, without imparting polypeptide content. This is achieved by lowering the pH value of the solution to a value below pH 6 and incubation the solution at this pH value.

BACKGROUND OF THE INVENTION

Biological macromolecules such as recombinant monoclonal antibodies and other proteins are used in a wide array of diagnostic and therapeutic areas. Especially monoclonal antibodies are now widely used in various severe diseases like cancer or rheumatoid arthritis. Commonly, these complex biological molecules are produced by fermentation processes with bacteria, yeasts or mammalian cells. Traditionally, the microbial or mammalian cells are removed from the fermentation broth by centrifugation or filtration and thereafter the cell free supernatant is further purified from fermentation related impurities by various methods like filtration, precipitation and chromatography.

Main impurities from the fermentation processes, beside the media components, are the residual amounts of nucleic acids (host cell DNA (HCDNA) and RNA) and Host Cell Proteins (HCP) from the cells that produce the biological macromolecule. For therapeutic purposes the acceptable concentrations of Host Cell DNA (HCDNA) or Host Cell Protein in the final drug substance are very low in order to reduce adverse effects and guarantee patient safety. Recent improvements in fermentation technology have led to higher cell densities in the production bioreactors, which increase the levels of HCP and HCDNA in the cell free supernatant placing higher demands on the purification process.

Effective and cheap methods to remove large amounts of HCDNA and HCP from cell culture supernatants are therefore highly desirable.

O'Brien, W. D., et al. (J. Acoust. Soc. Am. 52 (1972) 1251-1255) report the ultrasonic investigation of aqueous solutions of deoxyribose nucleic acid. Vorlickova, M., et al. (Nucl. Acids Res. 27(1999) 581-586) report the dimerization of the guanine-adenine repeat strands of DNA. Acid precipitation of mammalian cell fermentation broth is reported by Lydersen et al. (Lydersen, B. K., et al., Ann. N.Y. Acad. Sci. 745 (1994) 222-231). A method of isolating biomacromolecules using low pH and divalent cations is reported in WO 2008/127305. In EP 1 561 756 and EP 1 380 589 methods for purifying protein are reported.

SUMMARY OF THE INVENTION

Herein is reported a method for purifying a polypeptide from cell cultivation supernatants. It has been found that by adjusting the pH value in the acid range and subsequent incubation of the acidified solution for a specified time host cell nucleic acid and host cell protein precipitate but the target polypeptide remains in solution. Thereafter the precipitate and therewith the contaminating host cell components can be removed by a simple physical separation step. During the treatment of the solution the polypeptide of interest remains in solution whereas host cell contaminants are precipitated.

One aspect as reported herein is a method for producing or obtaining an immunoglobulin comprising the following steps:

-   -   i) adding a solution consisting of an acid and water to a cell         cultivation supernatant from which cells and cell debris have         been removed for adjusting the pH value to a value of from pH         4.5 to pH 5.5, whereby the solution is essentially free of         divalent cations,     -   ii) incubating the pH adjusted cell cultivation supernatant, and     -   iii) removing the precipitate from the incubated cell         cultivation supernatant and thereby producing or obtaining the         immunoglobulin,     -   wherein the cell cultivation supernatant comprises the         immunoglobulin at a concentration of not more than 10 mg/ml,     -   wherein the concentration of the added acid is 5.5 mol/l or         lower.

In one embodiment at least 90% of the immunoglobulin remains in solution during the incubating step. In a further embodiment at least 95% of the immunoglobulin remains in solution during the incubating step. In also an embodiment more than 98% of the immunoglobulin remains in solution during the incubating step.

In one embodiment the method for producing a polypeptide comprises the following steps:

-   -   a) cultivating a cell comprising a nucleic acid encoding the         polypeptide,     -   b) removing cells and cell debris from the cell cultivation         supernatant,     -   c) adjusting the pH value of the cell cultivation supernatant to         a value of below pH 5.5,     -   d) incubating the pH adjusted cell cultivation supernatant, and     -   e) removing the precipitate from the incubated cell cultivation         supernatant and thereby producing the polypeptide.

Also an aspect as reported herein is a method for purifying a cell cultivation supernatant comprising the following steps:

-   -   a) adjusting the pH value of the cell cultivation supernatant to         a value of below pH 5.5,     -   b) incubating the pH adjusted cell cultivation supernatant, and     -   c) removing the precipitate from the incubated cell cultivation         supernatant and thereby purifying the cell cultivation         supernatant.

Another aspect is a method for producing a polypeptide comprising the following steps:

-   -   i) providing a mammalian cell comprising a nucleic acid encoding         the polypeptide,     -   ii) cultivating the cell under serum free conditions,     -   iii) recovering the cell cultivation supernatant, optionally         removing cells and cell debris from the cell cultivation         supernatant, and     -   iv) purifying the cell cultivation supernatant by the following         steps and thereby producing a polypeptide:         -   a) adding a solution consisting of an acid and water to the             cell cultivation supernatant, which solution is essentially             free of divalent cations, for adjusting the pH value to a             value below pH 5.5,         -   b) incubating the pH adjusted cell cultivation supernatant,             and         -   c) removing the precipitate from the incubated cell             cultivation supernatant,     -   wherein the cell cultivation supernatant comprises the         polypeptide at a concentration of not more than 10 mg/ml,     -   wherein the concentration of the added acid is 5.5 mol/l or         lower.

In one embodiment adjusting the pH value is to a pH value of from pH 5.5 to pH 3.5. In a further embodiment adjusting the pH value is to a pH value of from pH 5.5 to pH 4.5.

In one embodiment incubating of the pH adjusted cell cultivation supernatant is at a temperature of from 1° C. to 30° C. In another embodiment incubating of the pH adjusted cell cultivation supernatant is at a temperature of from 2° C. to 10° C. In a further embodiment the incubating of the pH adjusted cell cultivation supernatant is at a temperature of about 4° C.

In one embodiment incubating of the pH adjusted cell cultivation supernatant is for about 0.5 hours or more. In another embodiment incubating of the pH adjusted cell cultivation supernatant is for about 0.5 hours to about 72 hours. In a further embodiment incubating of the pH adjusted cell cultivation supernatant is for about 2 hours to about 48 hours. In still another embodiment incubating of the pH adjusted cell cultivation supernatant is for about 20 hours to about 32 hours. In also another embodiment incubating the pH adjusted cell cultivation supernatant is for about 24 hours.

In one embodiment removing cells and cell debris and/or removing of the precipitate is by filtration, settlement, or centrifugation.

In another embodiment the cell cultivation supernatant is a mammalian cell cultivation supernatant.

In one embodiment the polypeptide is an immunoglobulin. In another embodiment the immunoglobulin is an immunoglobulin of the class G. In still another embodiment the immunoglobulin is of the class G subclass IgG1 or subclass IgG4 or variants thereof.

In one embodiment the immunoglobulin is of subclass IgG1 and the incubating is for about 2 hours to about 48 hours at a pH value of from pH 4.5 to pH 3.5.

In one embodiment the immunoglobulin is of subclass IgG4 and the incubating is for about 2 hours to about 30 hours at a pH value of from pH 5.5 to pH 4.5.

DETAILED DESCRIPTION OF THE INVENTION

Herein is reported a method for purifying a polypeptide comprising the following steps:

-   -   a) adjusting the pH value of a cell cultivation supernatant         comprising the polypeptide to a pH value of from pH 3.5 to 5.5,     -   b) incubating the pH adjusted cell cultivation supernatant, and     -   c) removing the precipitate from the incubated cell cultivation         supernatant and thereby purifying the polypeptide.

The method as reported herein can be performed directly with the crude cell fermentation supernatant from which cells and cell debris have been removed but also after one or more preliminary purification steps, such as protein A affinity chromatography.

The term “cell cultivation supernatant” denotes a solution that is obtained by the cultivation of a cell secreting a polypeptide of interest. The supernatant comprises beside the secreted polypeptide also components of the employed cell cultivation medium and metabolic components beside the polypeptide of interest secreted by the cells during the cultivation as well as other components of the cultivated cells set free from dead cells during the cultivation or from disintegrated cells during the recovery of the polypeptide from the cells. The cell cultivation supernatant is free of cell debris and/or intact cells. The term also includes a cell cultivation supernatant that has been processed by a single chromatographic purification step, such as protein A affinity chromatography.

The term “polypeptide” denotes a polymer consisting of amino acids joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 20 amino acid residues may be referred to as “peptides”, whereas molecules consisting of two or more polypeptides or comprising one polypeptide of more than 100 amino acid residues may be referred to as “proteins”. A polypeptide may also comprise non-amino acid components, such as carbohydrate groups, metal ions, or carboxylic acid esters. The non-amino acid components may be added by the cell, in which the polypeptide is expressed, and may vary with the type of cell. Polypeptides are defined herein in terms of their amino acid backbone structure or the nucleic acid encoding the same. Additions such as carbohydrate groups are generally not specified, but may be present nonetheless. In one embodiment of the method as reported herein is the polypeptide selected from immunoglobulins, immunoglobulin fragments and immunoglobulin conjugates.

The term “immunoglobulin” denotes a protein consisting of two or more polypeptide(s) substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include the different constant region genes as well as the myriad immunoglobulin variable region genes. An immunoglobulin in general comprises two so called light chain polypeptides (light chain) and two so called heavy chain polypeptides (heavy chain). Each of the heavy and light chain polypeptides contains a variable domain (variable region) (generally the amino terminal portion of the polypeptide chain) comprising binding regions that are able to interact with an antigen. Each of the heavy and light chain polypeptides comprises a constant region (generally the carboxyl terminal portion). The variable domain of an immunoglobulin's light or heavy chain comprises different segments, i.e. four framework regions (FR) and three hypervariable regions (CDR). The constant region of an immunoglobulin is not involved directly in binding to the immunoglobulin's antigen, but exhibits various effector functions. Depending on the amino acid sequence of the constant region of the heavy chains, immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG, and IgM. Some of these classes are further divided into subclasses (isotypes), i.e. IgG in IgG1, IgG2, IgG3, and IgG4, or IgA in IgA1 and IgA2. According to the class to which an immunoglobulin belongs the heavy chain constant regions of immunoglobulins are called α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM), respectively.

The term “immunoglobulin conjugate” denotes a polypeptide comprising at least one domain of an immunoglobulin heavy or light chain conjugated via a peptide bond to a further polypeptide. The further polypeptide is a non-immunoglobulin peptide, such as a hormone, or growth receptor, or cell-toxic peptide, or complement factor, or the like.

In one embodiment the immunoglobulin is an immunoglobulin of the class G. In another embodiment the immunoglobulin is of the class G subclass IgG1 or subclass IgG4 or a variant thereof. The term “variant” denotes a polypeptide that differs in the amino acid sequence from a parent polypeptide's amino acid sequence by virtue of addition, deletion and/or substitution of one or more amino acid residue(s) in the parent polypeptide amino acid sequence. In one embodiment a variant will have an amino acid sequence having at least 90% amino acid sequence identity with the parent polypeptides amino acid sequence, in another embodiment at least 95%, and in a further embodiment at least 99% amino acid sequence identity.

The term “adjusting the pH value” as used herein denotes the addition of an acid to a solution, especially to a cell cultivation supernatant, in order to lower the pH value of the solution to a pH value below pH 7.0. The adjusting can be achieved by the addition of an acidic solution, i.e. of an acid. In one embodiment the adjusting is by the addition of an acidic solution selected from hydrochloric acid, phosphoric acid, acetic acid, and citric acid.

The term “incubating” as used herein denotes that the respective solution is maintained at a set pH value. The incubating can be for a specific time. In one embodiment the incubating is for about 0.5 hours or more. In another embodiment the incubating is for about 0.5 hours to about 72 hours. In a further embodiment the incubating is for about 2 hours to about 48 hours. In still another embodiment the incubating is for about 20 hours to about 32 hours. In also another embodiment the incubating is for about 24 hours. In one embodiment the incubating is for a specific time as defined in the before listed embodiments at a pH value of from pH 3.5 to pH 5.5, especially at a pH value of from pH 4.5 to pH 5.5.

The term “about” as used herein denotes that the directly successive value is no exact value but rather denotes a range. This range is in one embodiment plus or minus 20% of the value, in another embodiment plus or minus 10% of the value and in a further embodiment plus or minus 5% of the value. For example, the term “about 24 hours” denotes the range of from 19.2 hours to 28.8 hours.

The term “essentially free” denotes that to a solution no such compound is added. But the specific compound may be present in minor amounts due to its presence in other compounds comprised in the solution. Generally a solution is essentially free of a compound when this compound is present in the solution at a concentration of 1 mM or less, especially at a concentration of 1 μM or less.

Methods for production of polypeptides are known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the polypeptide and usually purification to a pharmaceutically acceptable purity. For example, for the expression of an immunoglobulin in a cell, nucleic acids encoding the respective light and heavy chains are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells like CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6(R) cells, yeast, or E. coli cells, and the immunoglobulin is recovered from the cells (supernatant or cells after lysis).

The term “cell” as used in the current application denotes any kind of cellular system which can be engineered to produce a polypeptide. In one embodiment the cell is a mammalian cell. In another embodiment the cell is selected from HEK cells and CHO cells.

It has been found that nucleic acid and cell protein of a cultivated cell do precipitate but the secreted polypeptide remains in solution if the pH value of the cell cultivation supernatant is adjusted to a value below pH 5.5 and subsequently the solution is incubated for a specified time at that pH value. Thereafter the precipitate and therewith the contaminating host cell components can be removed by a simple physical separation step.

The method as reported herein can be used for any kind of cell cultivation supernatant, i.e. for eukaryotic cell cultivation supernatant and prokaryotic cell cultivation supernatants. In one embodiment the cell cultivation supernatant is a eukaryotic cell cultivation supernatant. In a further embodiment the supernatant is a mammalian cell cultivation supernatant. In another embodiment the supernatant is a CHO, HEK or Sp2/0 cell cultivation supernatant.

Today almost all cell cultivation processes are performed in the absence of added animal serum. Thus, in one embodiment the cell cultivation supernatant is obtained from a cultivation of the cell under serum free conditions. Additionally with the absence of added animal serum potential interference from compounds not known which are present in the animal serum are excluded and also the concentration of polypeptides in the cell cultivation supernatant is reduced. This is advantageous as generally in methods exploiting precipitation for purification purposes the co-precipitation of the polypeptide of interest can occur and therewith a reduction in yield of the purification process. The same effect can be observed in the presence of the cultivated cell or cell debris therefrom. Therefore, in one embodiment cells and cell debris is removed from the cell cultivation supernatant prior to the adjusting of the pH value.

The concentration of the polypeptide to be purified in the method as reported herein is not more than 10 mg/ml. In one embodiment the concentration of the polypeptide in the cell cultivation supernatant is of from 0.1 mg/ml to about 10 mg/ml. In another embodiment the concentration of the polypeptide in the cell cultivation supernatant is of from 1 mg/ml to 8 mg/ml. In a further embodiment the concentration of the polypeptide in the cell cultivation supernatant is of from 1 mg/ml to 5 mg/ml.

In order to adjust the pH value of the cell cultivation supernatant an acid has to be added to the cell cultivation supernatant. This acid may be any acid as long as that acid does not irreversibly interact with the polypeptide. In one embodiment the acid is selected from hydrochloric acid, phosphoric acid, sulphuric acid, formic acid, acetic acid, propionic acid, malonic acid, succinic acid, adipic acid, lactic acid, and citric acid. It has to be pointed out that the acid when added to the cell cultivation supernatant is an aqueous solution. The solution is consisting of the respective acid as free acid and water and is essentially free of other substances, especially divalent cations. The term “free acid” denotes that the acid is present in a form in which the acidic hydrogen atoms are present and not e.g. exchanged for a different cation.

This includes that the form of the acid used to prepare the solution is also the free acid; likewise it is excluded that the acid is used in form of a salt. In one embodiment is the acid selected from hydrochloric acid, phosphoric acid, acetic acid, and citric acid. The concentration of the acid in the respective solution is in one embodiment 5.5 mol/l or less. In another embodiment the concentration of the acid is of from 1 mol/l to 5.5 mol/l. In a further embodiment the concentration of the acid is of from 1.5 mol/l to 4 mol/l. Alternatively the concentration of the acid in the respective solution is in one embodiment 30 wt-% or less. In one embodiment the concentration of the acid is of from 1 wt-% to 30 wt-%. In another embodiment the concentration is of from 5 wt-% to 25 wt-%. In a further embodiment is the concentration of the acid of from 10 wt-% to 20 wt-%.

In general if herein ranges of values are given the range does expressly include the boundary points as listed.

After the adjustment of the pH value the pH adjusted cell cultivation supernatant is incubated for a specified time. This specific time is in one embodiment at least about 0.5 hours or more. In another embodiment the specific time is of from about 0.5 hours to about 72 hours. In a further embodiment specific time is of from about 2 hours to about 48 hours. In still another embodiment the specific time is of from about 20 hours to about 32 hours. In also another embodiment the specific time is about 24 hours. In one embodiment incubating the pH adjusted cell cultivation supernatant is at a temperature of from 1° C. to 30° C. In another embodiment incubating the pH adjusted cell cultivation supernatant is at a temperature of from 2° C. to 10° C. In a further embodiment incubating the pH adjusted cell cultivation supernatant is at a temperature of about 4° C.

With a method as reported herein host cell nucleic acid and host cell protein can be precipitated, i.e removed, from a cell culture supernatant without reducing the concentration of the produced polypeptide. An incubation time of about two hours can be sufficient to remove a large amount of host cell nucleic acid. Thus, in one embodiment the incubating is for about two hours. For the precipitation of host cell protein an incubation time of from about two hours to about 48 hours can be sufficient depending on the adjusted pH value and the polypeptide. Thus, in one embodiment the incubating is for about 2 hours at a pH value of about pH 5 at a temperature of about 4° C. In another embodiment the incubating is for about 2 hours to about 48 hours at a pH value of about 5 at a temperature of about 4° C.

In one embodiment the polypeptide is an immunoglobulin.

In one embodiment the immunoglobulin is an immunoglobulin of subclass IgG1. In another embodiment the incubating is for 2 hours to 48 hours at a pH value of from pH 4.5 to pH 3.5 and the immunoglobulin is an immunoglobulin of the subclass IgG1. In a specific embodiment the incubating is for 2 hours to 30 hours.

In one embodiment the immunoglobulin is an immunoglobulin of subclass IgG4. In another embodiment the incubating is for 2 hours to 48 hours at a pH value of from pH 5.5 to pH 4.5 and the immunoglobulin is an immunoglobulin of the subclass IgG4. In a specific embodiment the incubating is at a pH value of about pH 5.

After the incubating the precipitate has to be removed. For the removal any method known to a person of skill in the art can be used. Exemplary methods are filtration, sedimentation and decantation, centrifugation and settlement. In one embodiment the removing of the precipitate is by a method selected from sedimentation, filtration, settlement and centrifugation.

After the removal of the precipitate a further purification of the polypeptide can be performed, e.g. with chromatographic methods known to a person of skill in the art. Therefore in one embodiment the method as reported herein comprises the step of purifying the polypeptide with one or more chromatographic steps.

For the purification of immunoglobulins a combination of different column chromatography steps can be employed. In one embodiment a protein A affinity chromatography is followed by one or two additional chromatographic separation steps, e.g. ion exchange chromatographic steps. Different methods are well established and widespread used for protein recovery and purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis).

The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 Host cell DNA content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein.

FIG. 2 Host cell protein content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein.

FIG. 3 Antibody content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein.

FIG. 4 Host cell DNA content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein.

FIG. 5 Host cell protein content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein.

FIG. 6 Antibody content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein.

FIG. 7 Host cell DNA content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein.

FIG. 8 Host cell protein content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein.

FIG. 9 Antibody content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein.

FIG. 10 Antibody content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein depending on the pH value and the incubation time.

FIG. 11 Antibody content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein depending on the pH value and the incubation time.

FIG. 12 Antibody content in an anti-HER3 antibody fermentation culture supernatant obtained with a method as reported herein depending on the pH value and the incubation time.

EXAMPLE 1

Materials and Methods

Antibody

The herein reported method is exemplified with an anti-EGFR antibody as reported in WO 2008/017963.

Another exemplary immunoglobulin is an anti-PLGF antibody as reported in WO 2006/099698.

Another exemplary immunoglobulin is an anti-P-selectin antibody as reported in WO 2005/100402.

Another exemplary immunoglobulin is an anti-HER3 antibody as reported in PCT/EP2010/070062.

DNA

The residual DNA concentration was measured by Q-PCR (quantitative polymerase chain reaction). Therefore, the samples were incubated at high temperatures to denature the DNA in the sample. The DNA was captured from solution by a Silica matrix and eluted therefrom with buffer according to manufacturer's instructions. For the extraction a QIAcube robot was used including the QIAamp Viral RNA Kit (both from Qiagen, Hilden, Germany). Therefore the sample, lysis buffer and carrier RNA were combined and incubated for 10 minutes. Ethanol was added to each tube and the column of the QIAamp Viral RNA Kit was loaded with the sample-ethanol solution and centrifuged. Afterwards a wash solution was applied to the columns and again the column was centrifuged. Thereafter a different wash solution was applied to the column and the column was centrifuged again. After the elution buffer was applied the column is centrifuged twice.

For the quantification of the DNA a LightCycler 2.0 is used (Roche Diagnostics GmbH, Mannheim, Germany). In Table 1 the PCR parameters are outlined.

TABLE 1 PCR parameters. cycles step analysis mode temperature hold time 1 preincubation none 40° C. 10 min. 95° C. 10 min. 45 amplification quantification 95° C. 10 min. 60° C. 30 sec. 69° C. 1 sec. 1 cooling none 37° C. 30 sec.

During the procedure a DNA strands marked with dye binds to the DNA single strands. During the amplification the fluorescence increases proportional to the quantity of DNA.

HCP

The walls of the wells of a micro titer plate are coated with a mixture of serum albumin and Streptavidin. A goat derived polyclonal antibody against HCP is bound to the walls of the wells of the micro titer plate. After a washing step different wells of the micro titer plate are incubated with a HCP calibration sequence of different concentrations and sample solution. After the incubation not bound sample material is removed by washing with buffer solution. For the detection the wells are incubated with an antibody peroxidase conjugate to detect bound host cell protein. The fixed peroxidase activity is detected by incubation with ABTS and detection at 405 nm.

SEC

The chromatography was conducted with a Tosoh Haas TSK 3000 SWXL column on an ASI-100 HPLC system (Dionex, Idstein, Germany). The elution peaks were monitored at 280 nm by a UV diode array detector (Dionex). After dissolution of the concentrated samples to 1 mg/ml the column was washed with a buffer consisting of 200 mM potassium dihydrogen phosphate and 250 mM potassium chloride pH 7.0 until a stable baseline was achieved. The analyzing runs were performed under isocratic conditions using a flow rate of 0.5 ml/min. over 30 minutes at room temperature. The chromatograms were integrated manually with Chromeleon (Dionex, Idstein, Germany).

IEC

With the Ion Exchange Chromatography the charge heterogeneity of the protein was analyzed. A cation exchange Dionex ProPac chromatography column was used on a Dionex Chromeleon HPLC system.

Protein Determination

A chromatographic method was used to quantify the amount of antibody present in a sample. A Poros A column was used that binds the Fc-region of the antibody. The antibody binds to the column and is subsequently eluted by low pH conditions. Protein concentration was determined by determining the optical density (OD) at 280 nm, with a reference wavelength of 320 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.

EXAMPLE 2

Procedure

The samples were obtained directly from the cultivation medium of the respective antibody secreting cell lines. After removal of cells and cell debris the cultivation supernatant was divided into several aliquots of 200 ml each and adjusted to a pH value of pH 4.0, pH 5.0, and pH 6.0, respectively by adding 10% or 20% (v/v) acetic acid. In the reference aliquot the pH value was adjusted if required to approximately pH 7. The aliquots were stored at 4° C. and samples were taken from each aliquot after 2, 24 and 48 hours.

During storage different levels of precipitate formation were observed at the different pH values. The precipitate was removed by sedimentation. Alternatively, the precipitate can be separated by filtration or centrifugation.

After removal of the precipitate the clear liquid phase was analyzed for host cell DNA content, host cell protein (HCP) content and protein amount.

TABLE 2 Host cell DNA content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein. host cell DNA [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 7.36 7.02 3.19 8.30 24 7.63 6.59 2.30 8.88 48 7.71 6.95 7.31 6.74

TABLE 3 Host cell protein content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein. host cell protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 528 432 343 457 24 477 471 318 334 48 489 467 426 343

TABLE 4 Antibody content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein. protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 2268 2307 2207 1530 24 2332 2305 2205 1521 48 2321 2316 2231 1572

TABLE 5 Host cell DNA content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (1^(st) data set). host cell DNA [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 9.45 7.81 3.11 7.81 24 9.61 8.16 2.30 8.26 48 9.93 8.07 0.84 7.44

TABLE 5a Host cell DNA content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (2^(nd) data set). host cell DNA [μg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 5543 3007 286 846 24 2750 1828 65 58 48 3484 1504 103 49

TABLE 5b Host cell DNA content in the sediment of an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (2^(nd) data set). host cell DNA [μg/g] time [h] pH 5.0 pH 4.0 2 85395 223104 24 183908 242948 48 145992 229317

TABLE 6 Host cell protein content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (1^(st) data set). host cell protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 1258 1176 276 390 24 1197 1151 205 363 48 1297 1069 178 357

TABLE 6a Host cell protein content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (2^(nd) data set). host cell protein [mg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 983 1051 787 292 24 784 869 501 209 48 831 874 503 210

TABLE 6b Host cell protein content in the sediment of an anti- PLGF antibody fermentation culture supernatant obtained with a method as reported herein (2^(nd) data set). host cell protein [mg/g] time [h] pH 5.0 pH 4.0 2 1693 1299 24 2236 1667 48 2024 1021

TABLE 7 Antibody content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (1^(st) data set). protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 1410 1393 1360 644 24 1410 1395 1358 603 48 1410 1394 1356 567

TABLE 7a Antibody content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (2^(nd) data set). protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 1003 1051 1012 1023 24 1022 1045 1023 1067 48 1016 1044 1045 1045

TABLE 8 Host cell DNA content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (1^(st) data set). host cell DNA [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 11.38 5.85 2.55 13.35 24 11.52 5.94 1.31 6.85 48 11.22 5.69 1.66 87.40

TABLE 8a Host cell DNA content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (2^(nd) data set). host cell DNA [μg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 22068 10228 1 404 24 18250 7953 74 93 48 19305 6280 46 275

TABLE 8b Host cell DNA content in the sediment of an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (2^(nd) data set). host cell DNA [μg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 — — 65945 79413 24 — — 90656 41701 48 393649 130806 10126 159270

TABLE 9 Host cell protein content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (1^(st) data set). host cell protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 218 209 212 281 24 244 384 208 215 48 214 254 144 1877

TABLE 9a Host cell protein content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (2^(nd) data set). host cell protein [mg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 181 192 120 46 24 212 199 103 77 48 202 207 73 77

TABLE 9b Host cell protein content in the sediment of an anti- P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (2^(nd) data set). host cell protein [mg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 — — 290 367 24 — — 693 129 48 110 108 279 365

TABLE 10 Antibody content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (1^(st) data set). protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 1880 1880 1859 1094 24 1884 1885 1867 1399 48 1890 1881 1865 127

TABLE 10a Antibody content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (2^(nd) data set). protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 2950 2933 2929 2231 24 2948 2867 2900 2157 48 3051 2981 2978 2077

TABLE 11 Host cell DNA content in an anti-HER3 antibody fermentation culture supernatant obtained with a method as reported herein. host cell DNA [μg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 12333 15716 745 65 24 8053 19124 201 14 48 7703 15473 52 13

TABLE 11a Host cell DNA content in the sediment of an anti- HER3 antibody fermentation culture supernatant obtained with a method as reported herein. host cell DNA [μg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 — — 61749 57288 24 — — 67944 48404 48 304107 186101 71909 25617

TABLE 12 Host cell protein content in an anti-HER3 antibody fermentation culture supernatant obtained with a method as reported herein. host cell protein [mg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 522 432 186 40 24 488 384 141 31 48 476 346 115 87

TABLE 12a Host cell protein content in the sediment of an anti-HER3 antibody fermentation culture supernatant obtained with a method as reported herein. host cell protein [mg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 — — 770 465 24 — — 612 424 48 1167 986 726 275

TABLE 13 Antibody content in an anti-HER3 antibody fermentation culture supernatant obtained with a method as reported herein. protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 2992 3035 3047 2928 24 3005 3038 2997 2989 48 3012 3044 3003 2625 

1. A method for producing an immunoglobulin comprising the following steps: i) adding a solution consisting of an acid and water to a cell cultivation supernatant from which cells and cell debris have been removed for adjusting the pH value to a value of from pH 4.5 to pH 5.5, whereby the solution is essentially free of divalent cations, ii) incubating the pH adjusted cell cultivation supernatant, and iii) removing the precipitate from the incubated cell cultivation supernatant and thereby producing the immunoglobulin, wherein the cell cultivation supernatant comprises the immunoglobulin at a concentration of not more than 10 mg/ml, wherein the concentration of the added acid is 5.5 mol/l or lower.
 2. The method according to claim 1, wherein at least 90% of the immunoglobulin remains in solution during the incubating step.
 3. The method according to claim 1, wherein the incubating is at a temperature of from 2° C. to 10° C.
 4. The method according to claim 3, wherein the incubating of the pH adjusted cell cultivation supernatant is at a temperature of about 4° C.
 5. The method according to claim 1, wherein the incubating the pH adjusted supernatant is for about at least 2 hours.
 6. The method according to claim 5, wherein the incubating is for about 2 hours to about 72 hours.
 7. The method according to claim 6, wherein the incubating is for about 2 hours to about 48 hours.
 8. The method according to claim 7, wherein the incubating is for about 24 hours.
 9. The method according to claim 1, wherein the immunoglobulin is of subclass IgG1 and the incubating is for about 2 hours to about 48 hours at a pH value of from pH 4.5 to pH 3.5.
 10. The method according to claim 1, wherein the immunoglobulin is of subclass IgG4 and the incubating is for about 2 hours to about 30 hours at a pH value of from pH 5.5 to pH 4.5.
 11. The method according to claim 1, wherein the removing is by a method selected from sedimentation, decantation, filtration, settlement, and centrifugation.
 12. The method according to claim 1, wherein the acid is selected from acetic acid, citric acid, hydrochloric acid, and phosphoric acid.
 13. The method according to claim 1, wherein the concentration of the acid is of from 1.5 mol/l to 5.5 mol/l.
 14. The method according to claim 12, wherein the acid is acetic acid with a concentration of from 1.5 mol/l to 4 mol/l.
 15. The method according to claim 1, wherein the concentration of the immunoglobulin is of from 1 mg/ml to 5 mg/ml.
 16. The method according to claim 1, wherein the cell is a CHO cell.
 17. The method according to claim 1, wherein the incubating is for about 2 hours at a pH value of about pH 5 or about pH 4 at a temperature of about 4° C.
 18. The method according to claim 1, wherein incubating is for about 2 hours to about 48 hours at a pH value of about pH 5 or of about pH 4 at a temperature of about 4° C. 